本文選題：植酸 + 植酸酶��； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：通常情況下,糧食中的肌醇和磷大多以植酸的狀態(tài)存在。以植酸鹽狀態(tài)存在的有機(jī)磷酸化合物被稱為植酸磷。動(dòng)物要吸收飼料中以植酸磷狀態(tài)存在的磷,只有將植酸磷催化成無(wú)機(jī)磷酸鹽的形式。植酸酶屬磷酸單酯水解酶,是最近幾年出現(xiàn)的一種新型綠色添加劑,它通過(guò)催化過(guò)程,將植酸以及植酸鹽催化成磷酸(或鹽)和肌醇。有很多實(shí)驗(yàn)研究證明,使用植酸酶作為飼料添加劑時(shí),磷的吸收率可被提高,無(wú)機(jī)磷的補(bǔ)充量被減少,同時(shí)動(dòng)物糞便中無(wú)機(jī)磷的比重也下降;在飼料中植酸酶的加入能有效的抑制機(jī)體內(nèi)的植酸與金屬離子和蛋白質(zhì)生成絡(luò)合物,提高多種微量元素和蛋白質(zhì)的利用率。天然來(lái)源的植酸酶普遍存在表達(dá)水平低、提取困難、成本高等問(wèn)題,這些問(wèn)題都導(dǎo)致其難以滿足現(xiàn)實(shí)生產(chǎn)的需要。所以構(gòu)建基因工程菌作為微型生物反應(yīng)器來(lái)實(shí)現(xiàn)異源高水平分泌表達(dá),成為解決這一問(wèn)題的關(guān)鍵。通過(guò)基因工程手段能夠解決天然植酸酶分泌量低及抗逆性和熱穩(wěn)定性差等問(wèn)題。利用巴斯德畢赤酵母作為生物反應(yīng)器誘導(dǎo)重組植酸酶表達(dá)的方法是最具有優(yōu)勢(shì)的,該方法操作簡(jiǎn)便、表達(dá)量大且適宜工業(yè)化生產(chǎn)。憑借微生物工程技術(shù)的不斷發(fā)展,對(duì)植酸酶結(jié)構(gòu)和功能的探索正逐漸的成熟與深化。本文對(duì)來(lái)源于Yersinia kristensenii的植酸酶基因YkAPPA進(jìn)行改造,并成功實(shí)現(xiàn)了在大腸桿菌(Escherichia coli)EG60和畢赤酵母(Pichia pastoris)GS-115中異源表達(dá),同時(shí)對(duì)其重組植酸酶相關(guān)酶學(xué)特性和動(dòng)力學(xué)參數(shù)等進(jìn)行了研究分析,主要研究結(jié)果如下:1.利用NCBI數(shù)據(jù)庫(kù)公布的來(lái)源于Yersinia kristensenii(基因登錄號(hào)為JQ394763.1)的植酸酶基因序列,按照畢赤酵母的密碼子偏愛(ài)性對(duì)目的基因進(jìn)行優(yōu)化,優(yōu)化前與優(yōu)化后的氨基酸序列完全一致。設(shè)計(jì)合成長(zhǎng)度為60bp的小片段引物序列,利用PTDS基因合成技術(shù)合成了新的重組植酸酶基因YkAPPAS,重組植酸酶基因全長(zhǎng)為1266bp,依據(jù)密碼子偏好性將GC含量調(diào)整至51.9%。本實(shí)驗(yàn)獲得的植酸酶YkAPPAS經(jīng)過(guò)對(duì)比后發(fā)現(xiàn),其氨基酸序列中含有植酸酶的最具保守的兩個(gè)區(qū)域:分別是與植酸酶底物結(jié)合位點(diǎn)RHG×R×P和酶的催化中心HD。將合成的目的基因與pMD18-T載體連接克隆及測(cè)序,隨后將測(cè)序成功的目的基因與表達(dá)載體相連,合成pYPX88-YkAPPAS載體。利用電擊法將載體導(dǎo)入酵母GS-115中,篩選具有高活性的轉(zhuǎn)化株,用終濃度為1%的甲醇誘導(dǎo)培養(yǎng),發(fā)酵上清液中酶的分泌量可達(dá)到106.73μg/mL,完成了重組植酸酶YkAPPAS在畢赤酵母中的高活性、高產(chǎn)量的表達(dá)。在重組植酸酶YkAPPAS基因的5’端添加了6個(gè)His標(biāo)記,所以利用硫酸銨分級(jí)沉淀法進(jìn)行蛋白的初步純化后再利用Ni離子親和層析分離出單一的植酸酶重組蛋白。聚丙烯酰氨凝膠電泳結(jié)果顯示,重組植酸酶YkAPPAS的蛋白分子量約為46kDa,實(shí)際結(jié)果與生物信息學(xué)預(yù)測(cè)理論值一致,說(shuō)明YkAPPAS幾乎沒(méi)有糖基化現(xiàn)象的發(fā)生。2.將純化后的重組植酸酶進(jìn)行酶學(xué)特性分析,研究結(jié)果顯示,其最適pH分別是pH4.5和pH6,pH3-pH12時(shí)的活性都在30%以上,最適溫度在45℃,在100℃處理5min后其活性為47.55%,90℃處理5min后其活性為56.75%。EDTA及Mn2+對(duì)酶的活性有一定的激活作用;Cu2+以及Al3+、Pb2+、Zn2+等對(duì)酶活有一定的抑制作用,尤其是SDS抑制力最強(qiáng)。綜上所述,本實(shí)驗(yàn)獲得的相關(guān)酶活數(shù)據(jù)為應(yīng)用基因工程和蛋白質(zhì)工程等方法解決植酸酶分泌量少、活性低等問(wèn)題提供了重要的借鑒和參考價(jià)值。
[Abstract]:Usually, in the grain of inositol and phytic acid phosphorus mostly exist in the state. Organic phosphate compounds exist in phytate state called phytate phosphorus. Animal feed to absorb Phytic Acid P state of phosphorus, only the catalytic phytate into inorganic phosphate form. Phytic acid enzyme is phosphoric monoester hydrolases. Is a kind of new green additives in recent years, it will be through catalytic process, phytic acid and phytate catalyzed into phosphoric acid (or salt) and inositol. There are a lot of experiments, the use of phytase as feed additives, phosphorus absorption rate can be improved, adding amount of inorganic phosphorus was reduced, while inorganic phosphorus the proportion of animal manure also decreased; phytic acid with metal ions and protein complexes within the body to effectively inhibit phytase added in the feed, improve the utilization ratio of trace elements and protein. Day However, the source of phytase widespread low expression extraction difficulty, high cost, all these problems make it difficult to meet the real needs of production. So the construction of genetic engineering bacteria as micro bioreactor to achieve a high level of heterologous expression, is the key to solve this problem. By means of genetic engineering can solve the problems of natural phytase the secretion and low resistance and poor thermal stability. As a bioreactor induced by recombinant phytase expression in Pichia pastoris by Pasteur is the most has the advantage of this method is simple, the expression quantity and is suitable for industrial production. With the continuous development of microbial engineering technology, to explore the structure and function of phytase is gradually mature with the deepening of YkAPPA. The phytase gene from Yersinia kristensenii was improved, and the successful implementation in Escherichia coli ( Escherichia coli) and EG60 (Pichia pastoris) in Pichia pastoris heterologous expression in GS-115, and the recombinant phytase enzymatic characteristics and kinetic parameters were analyzed, the main results are as follows: 1. using the NCBI database published from Yersinia kristensenii (GenBank accession No. JQ394763.1) of the phytase gene sequence, according to the purpose optimized gene of Pichia pastoris codon preference, optimized amino acid sequence and the optimized length is exactly the same. The design and synthesis of small fragment primers of 60BP, recombinant phytase gene YkAPPAS was prepared by the PTDS gene synthesis technology, the full-length recombinant phytase gene is 1266bp, based on the codon preference of the phytase YkAPPAS GC the content of adjustment to obtain 51.9%. this experiment by comparison found that two Contained Phytase in the amino acid sequence of the most conservative Area: are with phytase substrate binding site RHG * R * P and enzyme catalytic center HD. synthesis gene and cloned into pMD18-T vector cloning and sequencing, then gene sequencing and expression vector successfully connected to pYPX88-YkAPPAS vector synthesis. Using electroporation. The carrier is introduced into yeast GS-115, screening with high activity the transformants were cultured for 1%, with the final concentration of methanol induced secretion in the supernatant enzyme can reach 106.73 u g/mL, the recombinant phytase YkAPPAS in Pichia pastoris expressed high activity and high yield. The recombinant phytase YkAPPAS gene 5 'end with 6 His markers, so the use of ammonium sulfate fractionation of protein purified by Ni ion affinity chromatography to isolate a single recombinant phytase protein. Polyacrylamide gel electrophoresis showed that the recombinant phytase YkAPPAS egg White molecular weight is about 46kDa, the actual results and the theoretical value of bioinformatics prediction, indicates that.2. YkAPPAS almost no glycosylation phenomenon will be carried out after the purification of recombinant phytase enzyme characteristics analysis, the results of the study showed that the optimum pH is respectively pH4.5 and pH6, pH3-pH12 activity in more than 30%, the most optimum temperature at 45 degrees, 100 degrees in the treatment of 5min after its activity was 47.55%, 90 5min after the treatment to activate the activity of 56.75%.EDTA and Mn2+ on the activity of enzyme; Cu2+ and Al3+, Pb2+, Zn2+ has an inhibitory effect on the enzyme activity, especially SDS. To sum up the strongest inhibition in this experiment, enzymes related to live data for the application of gene engineering and protein engineering methods to solve the phytase secretion of small, low activity provides an important reference value.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;Q55

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